Calmodulin activation of adenylate cyclase in the mouse B16 melanoma

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Abstract
Calmodulin antagonists inhibited hormone-stimulated cAMP accumulation in both cultured cells and cell lysates of mouse B16 melanoma. Particulate preparations of B16 melanoma contained 34-45% of total cell calmodulin, which could not be dissociated by extensive washing irrespective of the presence of EGTA [ethyleneglycol bis(.beta.-aminoethyl ether)N,N,N''N''-tetraacetic acid] in the buffer. The adenylate cyclase activity in such preparations was unaffected by the addition of exogenous calmodulin. However, the rare earth metal ion La3+, which can mimic or replace Ca2+ in many systems, produced an immediate inhibition of agonist-stimulated adenylate cyclase activity and preincubation of particulate preparations with La3+ followed by washing with La3+-free buffer dissociated calmodulin (96% loss) from particulate preparations. The loss of calmodulin from particulate preparations was associated with a decrease in agonist responsiveness of (74%) and a marked change in the Ca2+-sensitivity of the enzyme, low concentrations of Ca (approximately 10 nM) now failing to stimulate enzyme activity, high concentrations of Ca (.gtoreq. 100 nM) producing greater-than-normal inhibition of enzyme activity. Direct activation of adenylate cyclase by the addition of pure calmodulin was now demonstrable in such calmodulin-depleted particulate preparations. Half-maximal stimulation of agonist-responsive adenylate cyclase occurred at 80 nM calmodulin in the presence of 10 .mu.M free Ca2+. Maximal stimulation by calmodulin (at 300-600 nM) restored enzyme activity to 89 .+-. 5% (mean .+-. standard error of the mean, n = 7) of the activity in untreated, calmodulin-intact, preparations.